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| United States Patent |
5,334,391
|
|
Clark
, et al.
|
August 2, 1994
|
Intracellularly cleavable compounds
Abstract
Intracellularly cleavable derivatives of toxic compounds including
antibiotics are described.
| Inventors:
|
Clark; Brian R. (Redwood City, CA);
Nag; Bishwajit (Pacifica, CA)
|
| Assignee:
|
Anergen, Inc. (Redwood City, CA)
|
| Appl. No.:
|
890187 |
| Filed:
|
May 29, 1992 |
| Current U.S. Class: |
424/450; 428/402.2 |
| Intern'l Class: |
A61K 009/127 |
| Field of Search: |
424/450
436/839
428/402.2
516/2,12,14,21
530/327,387
|
References Cited
U.S. Patent Documents
| 4460560 | Jul., 1984 | Tokes | 424/1.
|
| 4839175 | Jun., 1989 | Guo | 424/450.
|
| 4847240 | Jul., 1989 | Ryser | 514/12.
|
| 4861581 | Aug., 1989 | Epstein | 424/1.
|
| 4873088 | Oct., 1989 | Mayhew | 424/450.
|
Primary Examiner: Kishore; G. S.
Attorney, Agent or Firm: Townsend and Townsend Khourie and Crew
Parent Case Text
This is a division of application Ser. No. 07/523,334 filed May 14, 1990,
now a U.S. Pat. No. 5,169,934.
Claims
We claim:
1. A liposome having a compound anchored in the liposome bilayer said
compound having the formula:
##STR5##
in which L is a doxorubicin moiety and X has a value of from 11 to 17.
2. A liposome as defined by claim 1 in which X in the formula of said
compound has a value of 15.
3. A system for delivery of a cytocidal agent to a cell which system
comprises a liposome composition including a compound having the formula
of claim 1.
Description
FIELD OF THE INVENTION
This invention relates to novel, intracellularly cleavable, disulfide
linked derivatives of ligands, including particularly the anthracycline
glycoside antibiotics exemplified by doxorubicin (Adriamycin),
doxorubicinol, daunorubicin and daunorubicinol; to therapeutically active
compositions such as liposomes which include such derivatives and to
mammalian, including human, diagnostic and therapeutic procedures in which
such derivatives and compositions are utilized.
BACKGROUND OF THE INVENTION
Many drugs such as doxorubicin, a potent cancer therapeutic agent, and
Amphotericin B, the most effective drug presently known for a broad range
of fungal infections, are highly toxic.
Liposome systems have been proposed as bloodstream delivery systems to
provide controlled release and to minimize toxic side effects of a variety
of encapsulated drugs, such as doxorubicin and Amphotericin B.
Various limitations on intravenous liposome drug delivery have been
recognized. Importantly included among these limitations are unduly rapid
release or leakage of the encapsulated drug from the liposome and insipid
uptake of blood circulatory liposomes by the reticuloendothelial system
(RES) which comprises circulating and fixed macrophages.
SUMMARY OF THE INVENTION
This invention provides novel, all internalizable intracellularly cleavable
derivatives of diagnostically and therapeutically useful ligands. Such
derivatives are efficient bloodstream delivery systems for the cleavable
ligand. These derivatives may be parenterally administered as such or as
associated with liposomes or biodegradable microspheres.
In its broader aspects, the invention includes derivatives of releasable
ligands which may bind to any of a variety of targets. The ligand moieties
are joined to the balance of the novel derivatives of the invention by an
intracellularly cleavable disulfide linkage. The novel cell internalizable
derivatives of the invention may have the formula
##STR1##
in which L is or is included in an intracellularly releasable ligand
moiety and X is any organic radical. Preferably, X provides a function to
be internalized by or attached to cells. X may enable detection, modify
cellular function or serve some other diagnostic or therapeutic purpose.
The novel derivatives of the invention are prepared by known chemical
methods as will be apparent to those skilled in the art.
One important embodiment of the invention comprises novel disulfide linked
ligand-peptide derivatives. Another embodiment of the invention designed
for liposomal delivery of therapeutic or diagnostic agents includes
disulfide linked ligand-lipid derivatives which may be incorporated into
liposome bilayers by standard techniques. Such derivatives may be anchored
to the liposome bilayer by the lipid moiety.
DETAILED DESCRIPTION OF THE INVENTION
A. The Ligands
The nature of the ligands useful as moieties of the novel derivatives of
the invention is determined by the function that the ligand is to perform
after intracellular cleavage. In general, such ligands include, but are
not limited to, receptor agonists, receptor antagonists, antineoplastic
agents such as doxorubicin, peptides, poly and monoclonal antibodies,
polynucleic acids, antitoxins, antifungal agents and enzyme inhibitors.
The various cancer chemotherapeutic and antifungal ligands are described
in the relevant patents and publications. Any peptide may be utilized that
is a ligand for a natural receptor. Ligands useful to provide mucosal
tissue retention are known to the art. See U.S. Pat. No. 4,839,175.
Anthracycline glycoside ligands useful in the invention include
anthraquinone structures having one quinone and hydroquinone group on
adjacent rings of the anthracene ring structure. Two groups of
antineoplastic anthraquinones having these features are illustrated in
FIGS. 1 and 2. Many other compounds of this type are described in the
prior art.
Included in the FIG. 1 group are a number of clinically important
antineoplastic drugs, such as doxorubicin, daunomycin, carcinomycin,
N-acetyladriamycin, N-acetyldaunomycin, rubidasone, and 5-imidodaunomycin.
Table I below gives the structure variations of these several class I
drugs, in terms of the R.sub.1, R.sub.2 and R.sub.3 groups in FIG. 1.
TABLE I
__________________________________________________________________________
R.sub.1
R.sub.2 R.sub.3
__________________________________________________________________________
Adriamycin .dbd.O
--CO--CH.sub.2 OH
--NH.sub.2
Daunomycin .dbd.O
--CO--CH.sub.3
--NH.sub.2
N-Acetyladriamycin
.dbd.O
--CO--CH2OH --NH--CO--CH.sub.3
N-Acetyldaunomycin
.dbd.O
--CO--CH.sub.3
--NH--CO--CH.sub.3
Rubidazone .dbd.O
--C--N--NH--C--CH.sub.3 O
--NH.sub.2
5-Iminodaunomycin
.dbd.NH
--CO--CH.sub.3
--NH.sub.2
__________________________________________________________________________
Drugs in this class are known to have antineoplastic effects against a
variety of cancers, including acute leukemia, breast cancer, Hodgkin
disease, non-Hodgkin lymphomas and sarcomas.
A second group of anthracene glycosides, which are distinguished from the
class I compounds by more complex (multimeric) amino glycoside residues,
as seen in FIG. 2. These compounds share the same general therapeutic and
toxicity properties of their class I counterparts. Representative Class II
anthracene aminoglycosides are listed in Table II, with reference to the
R.sub.1, R.sub.2 and R.sub.3 groups shown in FIG. 2.
TABLE II
______________________________________
Anthracycline
R.sub.1 R.sub.2 R.sub.3
______________________________________
Musettamycin OH COOCH.sub.3
H
Rudolfomycin OH COOCH.sub.3
Rednosamine
Aclaciaomycin
H COOCH.sub.3
Cinerulose
Marcellomycin
OH COOCH.sub.3
2-Deoxyfucose
Descarbomethoxy-
OH H 2-Deoxyfucose
marcellomycin
Descarbomethoxy-
OH H Rednosamine
rudolfomycin
______________________________________
B. The Intracellularly Cleavable Compounds
Intracellularly releasable peptides are known. See, e.g., Truet, et al.
(1982) EORTC Symposium: "Promising New Anti-Cancer Agents in Clinical
Trials" (Mathe, G., Ed.). Masson, Paris; Shen, et al. (1981) Biochem.
Biophys. Res. Comm. 102:1048-1054; Blattler, et al. (1985) Biochem.
24:1517-1524; and Marsh, et al. (1987) J.Biol.Chem. in press.
Intracellular cleavage is imparted to the derivatives of this invention by
a disulfide linkage which joins the ligand to the balance of the molecule.
The chemistry for the production of such disulfide linkages is known.
Appropriate chemistry can be selected for use with any desired ligand
functionality. An appropriate functionality, e.g., a primary amine
(--NH.sub.2) substituent, an hydroxyl or a carboxyl may be provided in
known chemical manner to ligands which lack such functionality.
For the purposes of this invention, ligands which have or which have been
provided with amine, preferably primary amine, functionality are
preferred.
The ligand amine functionality may be reacted with 2,2'-dithiodipyridine
and 2'-iminothiolane in solution in dimethylacetamide to produce an
N-(4-pyridyldithiobutyrimido) derivative of the ligand pursuant to
Equation I in which L represents any ligand.
##STR2##
Preferably L-NH.sub.2 is an antineoplastic anthraquinone such as
doxorubicin of the kind shown by FIG. 1 and FIG. 2. L-NH.sub.2 may also be
amphotericin B or another antifungal agent having an --NH.sub.2
functionality.
The Compound A is utilized to produce a disulfide linked peptide or lipid
derivative of the ligand. For example, the peptide derivative may be
produced pursuant to Equation II by reacting Compound A with a
mercaptopeptide X-SH in which X is any peptide.
##STR3##
X is preferably a peptide having from about 5 to 50 residues. X may also be
an alkyl group R. R groups having from about 12 to 18 carbon atoms are
miscible in liposome bilayers and serve as anchors for minimizing linkage
when the derivatives of this invention are administered in the form of
liposome delivery systems. Toxic peptides are preferred for some
therapeutic uses.
When L or X is toxic, compounds having the formula of Compound B are toxic
to cells when internalized. The toxicity is apparently consequent from
cleavage of the disulfide link with consequent internal release of one or
both the toxic moieties L and X.
EXAMPLE I
Synthesis of a Disulfide-Linked Doxorubicin (Adriamycin)-Lipid Derivative
The reactions are similar to those described for the disulfide-linked
peptide-Adriamycin derivative. The Adriamycin intermediate described in
Example I is used:
##STR4##
The purified Adriamycin-lipid derivative is incorporated into liposomes by
standard techniques. See, generally, Pozansky, M. J., et al. (1984)
Pharmacol. Rev. 36:277-336. After uptake of the liposomes by cells, the
internalized derivative is reductively cleaved by reduced glutathione, and
the released, toxic Adriamycin is cytocidal.
C. The Liposomal Compositions of the Invention
Methods for preparing derivatives containing liposomes of this invention
generally follow conventional liposome preparation methods. In one
preferred method, vesicle forming lipids are taken up in a suitable
organic solvent or solvent system and dried in vacuum or in an inert gas
to a lipid film. The derivative is included in the lipids forming the
film. The concentration of the derivative in the lipid solution is
preferably in molar excess of the final maximum concentration of the drug
in the liposome. The dried lipid/drug film is hydrated with a
physiologically compatible medium, preferably physiological saline. The
lipids hydrate to form a suspension of multilamellar vesicles (MLVs) whose
size typically range from about 0.5 microns to at least about 10 microns.
In general, the size distribution of MLVs in the above procedures can be
shifted toward smaller sizes by hydrating the lipid film more rapidly,
with shaking.
The liposome suspension may be sized to achieve a selective size
distribution of vesicles in a size range less than about 1 micron and
preferably between about 0.05 to 0.5 microns, and most preferably between
about 0.005 and 0.2 microns. The sizing serves to eliminate larger
liposomes and to produce a defined size range having optimal
pharmacokinetic properties.
Several known techniques are available for reducing the sizes and size
heterogeneity of liposomes. Sonicating a liposome suspension either by
bath or probe sonication produces a progressive size reduction down to
small unilamellar vesicles (SUVs) less than about 0.05 microns in size. A
known sonicating procedure is preferably used in reducing liposome sizes
to about 0.2 microns or less. Homogenization is another method which
relies on shearing energy to fragment large liposomes into smaller ones.
In a typical homogenization procedures, MVLs are recirculated through a
standard emulsion homogeneizer until selected liposome sizes, typically
between about 0.1 and 0.5 microns, are observed. In both methods, the
particle size distribution can be monitored by conventional laser-beam
particle size discrimination.
Extrusion of liposomes through a small-pore polycarbonate membrane is an
effective method for reducing liposome sizes down to a relatively
well-defined size distribution whose average in the range between about
0.1 and 1 micron, depending on the pore size of the membrane. Typically,
the suspension is cycled through the membrane several times until the
desired liposome size distribution is achieved. The liposomes may be
extruded through successively smaller pore membranes, to achieve a gradual
reduction in liposome size.
Centrifugation and molecular size chromatography are other methods which
are available for producing a liposome suspension with particle sizes
below a selected threshold less than 1 micron: These two methods both
involve preferential removal of larger liposomes, rather than conversion
of large particles to smaller ones. Liposome yields are correspondingly
reduced.
D. The Therapeutic and Diagnostic Compositions
The invention importantly includes therapeutically and diagnostically
useful compositions including the novel intracellularly releasable
derivatives described herein. These derivatives may be administered to a
mammalian subject, including humans. The prescribing physician will
ultimately determine the appropriate dose for a given human subject. The
dose can be expected to vary according to the age, weight, and response of
the individual as well as the nature and severity of the patient's
symptoms.
The mode of administration may determine the sites and cells in the
organism to which the compound will be delivered. For instance, delivery
to a specific site of infection may be most easily accomplished by topical
application (if the infection is external, e.g., on areas such as eyes,
skin, in ears, or on afflictions such as wounds or burns) or by absorption
through epithelial or mucocutaneous linings (e.g., nasal, oral, vaginal,
rectal, gastrointestinal, mucosa, etc. ). Such topical application may be
in the form of creams or ointments. The liposome-entrapped materials can
be administered alone but will generally be administered in admixture with
a pharmaceutical carrier selected with regard to the intended route of
administration and standard pharmaceutical practice. Such materials may be
injected parenterally, for example, intravenously, intramuscularly, or
subcutaneously. For parenteral administration, such materials are best
used in the form of a sterile aqueous solution which may contain other
solutes, for example, enough salts or glucose to make the solution
isotonic.
For the oral mode of administration, compositions of this invention can be
used in the form of tablets, capsules, lozenges, troches, powders, syrups,
elixirs, aqueous solutions and suspensions, and the like. In the case of
tablets, carriers w which can be used include lactose, sodium citrate, and
salts of phosphoric acid. Various disintegrants such as starch, and
lubricating agents such as magnesium stearate, sodium lauryl sulfate and
talc, are commonly used in tablets. For oral administration in capsule
form, useful diluents are lactose and high molecular weight polyethylene
glycols. When aqueous suspensions are required for oral use, certain
sweetening and/or flavoring agents can be added.
The derivatives of this invention may also be used in diagnostic assays; in
this case the amount of the composition used will depend on the
sensitivity of the liposome-coupled derivative to the target components in
the sample.
Unilamellar and multilamellar liposomes formed in conventional manner are
useful in the invention. Vesicle forming lipids which generally include
neutral and negatively charged phospholipids and a sterol such as
cholesterol are appropriate. Vesicles comprising
dipalmitoylphosphatidylcholine (DPPC) or distearoylphosphatidylcholine
(DSPC) are preferred and may be prepared in known manner. See, e.g.,
Tomita, T., et al. (1989 ), Biochim. Biophys. Acta 978:185-190.
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